Purification of truncated and mutated Chemotaxis Inhibitory Protein of Staphylococcus aureus--an anti-inflammatory protein.
The Chemotaxis Inhibitory Protein of Staphylococcus aureus (CHIPS) binds and blocks the C5a receptor (C5aR) and formyl-peptide receptor (FPR). This way, CHIPS is a potent inhibitor of the immune cell recruitment associated with inflammation. Truncation of the protein and the introduction of mutations, shifts the expression towards the insoluble fraction of Escherichia coli, whereas the wild-type protein can be solubly expressed. A protocol for expression and tag independent purification of biologically active CHIPS variants has been established to enable further characterization of an improved CHIPS variant, called ADC-1004. The CHIPS variants were purified by washing of E. coli inclusion bodies followed by refolding and gel filtration. New techniques were utilized to optimize the purification process. Expression in inclusion bodies was increased by the use of Ultra Yield flasks and optimal refolding conditions were determined by the use of the iFOLD Refolding System 2. The folding and biological activity of the purified proteins were analyzed by circular dichroism (CD) spectroscopy and flow cytometry, respectively, and compared to solubly produced CHIPS(31-113) and wild-type CHIPS(1-121). We show that the CHIPS variants produced in inclusion bodies can be refolded and purified to achieve equal biological activity as solubly produced CHIPS(31-113) and wild-type CHIPS(1-121). The truncation causes minor structural changes while purification from inclusion bodies or the soluble fraction does not further affect the structure.
Gustafsson
Erika
E
Alligator Bioscience AB, Scheelevägen 19A. S-223 70 Lund, Sweden. erg@alligatorbioscience.com
Forsberg
Cecilia
C
Haraldsson
Karin
K
Lindman
Stina
S
Ljung
Lill
L
Furebring
Christina
C
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000302:isolation & purification* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D013211:Staphylococcus aureus
D000894:Anti-Inflammatory Agents, Non-Steroidal; D001426:Bacterial Proteins; C493134:chemotaxis inhibitory protein, Staphylococcus aureus